Coumarin-based compounds and related methods

Abstract

The present invention generally relates to coumarin-based compounds that can be used for the labeling of biological molecules, as well as related synthetic and testing methods. The coumarin-based compounds typically survive conditions necessary for automated synthesis of nucleic acids without undergoing any substantial degree of degradation or alteration.

The invention claimed is: 
     
       1. A compound of Structure  15 ,  16  or  17 : 
       
         
           
           
               
               
           
         
         where
 m is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; 
 n is 1,2 or 3; 
 o is 0, 1, 2, or 3; 
 p is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 
 R 2  is selected from a group consisting of —H or —CH 3 ; 
 X is —OH, —NH 2 , —SH, —OR 11  —NHR 12  —NR 13 R 14  —OC(O)R 15  —NHC(O)R 16  , SC(O)R 17  , —C(O)OH, —C(O)OR 18 , —C(O)NHR 19 , —C(O)NR 20 R 21 , —N 3 ; 
 R 11 , R 12 , R 13 , R 14 , R 15 , R 16 , R 17 , R 18 , R 19 , R 20 , and R 21 , are independently selected from (C 1 -C 8 )alkyl, (C 1 -C 8 )substituted alkyl, —CH 2 CH(OH)CH 2 (OH), —CH(CH 2 OH) 2 , —CH 2 CCH, —(CH 2 CH 2 O)a —CH 2 CH 2 OH where a is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, —P(NR 22 R 23 )OCH 2 CH 2 -EWG, —P(O)(O − )-Oligonucleotide, —(CH 2 ) b CC-Nucleoside, —(CH 2 ) b CC-[Nucleoside Triphosphate], —(CH 2 ) b -Nucleoside, —(CH 2 ) b -[Nucleoside Triphosphate], —(CH 2 ) b NH-Nucleoside, —(CH 2 ) b NH-[Nucleoside Triphosphate], —(CH 2 ) b NHC(O)(CH 2 ) b -Nucleoside, and —(CH 2 ) b NHC(O)(CH 2 ) b -[Nucleoside Triphosphate], where b is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; 
 EWG is an electron withdrawing group; and 
 R 22  and R 23  are independently selected from —CH 3 , —CH 2 CH 3 , —CH(CH 3 ) 2  or R 22  and R 23  together form a heterocycle; and 
 
       
       in the compound of Structure 15, R x  and R y  are independently H or CH 3 , 
       in the compound of Structure 16, R 7  and R 8  are independently selected from H, (C 1 -C 8 ) alkyl, (C 1 -C 8 )substituted alkyl, or R 7  and R 8  together form a five-, six-, or seven-membered heterocycle; and 
       in the compound of Structure 17, R 24  is (C 1 -C 8 )alkyl or (C 1 -C 8 )substituted alkyl; 
       R 25  is (C 1 -C 8 )alkyl, (C 1 -C 8 )substituted alkyl or CH 2 SO 3 H; and 
       the dashed line represents an optional double bond. 
     
     
       2. The compound according to  claim 1 , wherein m is 1, 2 or 3, n is 1 or 2, o is 1 or 2, and p is 0, 1, 2 or 3. 
     
     
       3. The compound according to  claim 1 , wherein X is —OH, —OR 11 , or OC(O)R 15 . 
     
     
       4. A compound of Structure 14: 
       
         
           
           
               
               
           
         
         where
 m is 0; 
 n is 1, 2 or 3; 
 o is 1, 2, or 3; 
 p is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 
 R 2  is selected from a group consisting of —H, or —CH 3 ; 
 R 3  is selected from a group consisting of —H, -halogen, —CF 3 , —OH, —NH 2 , —CCH, —CN, —NO 2 , (C 1 -C 8 )alkyl, (C 1 -C 8 )substituted alkyl, (C 1 -C 8 )alkylthio, (C 1 -C 8 ) substituted alkylthio, (C 1 -C 8 )alkoxy, (C 1 -C 8 )substituted alkoxy, —NR 7 R 8  where R 7  and R 8  are independently selected from H, (C 1 -C 8 )alkyl or R 7  and R 8  together form a four-, five-, six-, or seven-membered heterocycle, (C 1 -C 8 )heteroalkyl, (C 1 -C 8 )substituted heteroalkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, or R 3  together with R 4  form a five- or six-membered carbocyclic or heterocyclic ring or substituted heterocyclic ring; 
 R 4  is selected from a group consisting of —H, -halogen, —CF 3 , —OH, —NH 2 , —CCH, —CN, —NO 2 , (C 1 -C 8 )substituted alkyl, (C 1 -C 8 )alkylthio, (C 1 -C 8 ) substituted alkylthio, (C 1 -C 8 )alkoxy, (C 1 -C 8 )substituted alkoxy, —NR 7 R 8  where R 7  and R 8  are independently selected from H, (C 1 -C 8 )alkyl or R 7  and R 8 together form a four-, five-, six-, or seven-membered heterocycle, (C 1 -C 8 )heteroalkyl, (C 1 -C 8 )substituted heteroalkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, or R 4  together with R 5  form a five- or six-membered carbocyclic or heterocyclic ring having nitrogen as a heteroatom or substituted heterocyclic ring having nitrogen as a heteroatom; 
 R 5  is selected from a group consisting of —H, -halogen, —CF 3 , —OH, —NH 2 ,—CCH, —CN, —NO 2 , (C 1 -C 8 )alkyl, (C 1 -C 8 )substituted alkyl, (C 1 -C 8 )alkylthio, (C 1 -C 8 ) substituted alkylthio, (C 1 -C 8 )alkoxy, (C 1 -C 8 )substituted alkoxy, —NR 7 R 8  where R 7  and R 8  are independently selected from H, (C 1 -C 8 )alkyl or R 7  and R 8 together form a four-, five-, six-, or seven-membered heterocycle, (C 1 -C 8 )heteroalkyl, (C 1 -C 8 )substituted heteroalkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, or R 5  together with R 6  form a five- or six-membered carbocyclic or heterocyclic ring or substituted heterocyclic ring; or R 4 , R 5  and R 6  together form fused carbocyclic or heterocyclic ring systems or substituted heterocyclic ring systems having five- or six-membered rings; 
 R 6  is selected from a group consisting of —H, -halogen, —CF 3 , —OH, —NH 2 , —CCH, —CN, —NO 2 , (C 1 -C 8 )alkyl, (C 1 -C 8 )substituted alkyl, (C 1 -C 8 )alkylthio, (C 1 -C 8 ) substituted alkylthio, (C 1 -C 8 )alkoxy, (C 1 -C 8 )substituted alkoxy, —NR 7 R 8  where R 7  and R 8  are independently selected from H, (C 1 -C 8 )alkyl or R 7  and R 8  together form a four-, five-, six-, or seven-membered heterocycle, (C 1 -C 8 )heteroalkyl, (C 1 -C 8 )substituted heteroalkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl; 
 X is selected from —OH —NH 2  —SH —OR 11  —NHR 12  —NR 13 R 14  —OC(O)R 15  —NHC(O)R 16  —SC(O)R 17 , —C(O)OH, —C(O)OR 18 , —C(O)NHR 19 , —C(O)NR 20 R 21 , —N 3 ; 
 R 11 , R 12 , R 13 , R 14 , R 15 , R 16 , R 17 , R 18 , R 19 , R 20 , and R 21 , are independently selected from (C 1 -C 8 )alkyl, (C 1 -C 8 )substituted alkyl, —CH 2 CH(OH)CH 2 (OH), —CH(CH 2 OH) 2 , —CH 2 CCH, —(CH 2 CH 2 O)a—CH 2 CH 2 OH where a is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, —P(NR 22 R 23 )OCH 2 CH 2 -EWG, —P(O)(O − )-Oligonucleotide, —(CH 2 ) b CC-Nucleoside, —(CH 2 ) b CC -[Nucleoside Triphosphate], —(CH 2 ) b -Nucleoside, —(CH 2 ) b -[Nucleoside Triphosphate], —(CH 2 ) b NH-Nucleoside, —(CH 2 ) b NH-[Nucleoside Triphosphate], —(CH 2 ) b NHC(O)(CH 2 ) b -Nucleoside, —(CH 2 ) b NHC(O)(CH 2 ) b -[Nucleoside Triphosphate], where b is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; 
 EWG is an electron withdrawing group; and 
 R 22 , and R 23 , are independently selected from —CH 3 , —CH 2 CH 3 , —CH(CH 3 ) 2  or R 22  and R 23  together form a heterocycle. 
 
       
     
     
       5. The compound according to  claim 4 , wherein R 3  is —H or (C 1 -C 8 )alkyl. 
     
     
       6. The compound according to  claim 5 , wherein X is —OH, —OR 11 , or OC(O)R 15 . 
     
     
       7. The compound according to  claim 6 , wherein R 4  is —H or R 4  together with R 5  form a five- or six-membered carbocyclic or heterocyclic ring. 
     
     
       8. A compound of Structure 14: 
       
         
           
           
               
               
           
         
         where
 m is 0; 
 n is 0; 
 o is 0; 
 p is 3, 4, 5, 6, 7, 8, 9, or 10 
 R 2  is selected from a group consisting of —H, or —CH 3 ; 
 R 3  is selected from a group consisting of —H, -halogen; 
 R 4  is selected from a group consisting of —H, -halogen, —CF 3 , —OH, —NH 2 , —CCH, —CN, —NO 2 , (C 1 -C 8 )alkyl, (C 1 -C 8 )substituted alkyl, (C 1 C 8 )alkylthio, (C 1 -C 8 ) substituted alkylthio, (C 1 -C 8 )alkoxy, (C 1 C 8 )substituted alkoxy, -NR 7 R 8  where R 7  and R 8  are independently selected from H, (C 1 -C 8 )alkyl or R 7  and R 8 together form a four-, five-, six-, or seven-membered heterocycle, (C 1 -C 8 )heteroalkyl, (C 1 -C 8 )substituted heteroalkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, or R 4  together with R 5  form a five- or six-membered carbocyclic or heterocyclic ring having nitrogen as a heteroatom or substituted heterocyclic ring having nitrogen as a heteroatom; 
 R 5  is selected from a group consisting of —H, -halogen, —CF 3 , —OH, —NH 2 , —CCH, —CN, —NO 2 , (C 1 -C 8 )alkyl, (C 1 -C 8 )substituted alkyl, (C 1 C 8 )alkylthio, (C 1 -C 8 ) substituted alkylthio, (C 1 -C 8 )alkoxy, (C 1 C 8 )substituted alkoxy, —NR 7 R 8  where R 7  and R 8  are independently selected from H, (C 1 -C 8 )alkyl or R 7  and R 8 together form a four-, five-, six-, or seven-membered heterocycle, (C 1 -C 8 )heteroalkyl, (C 1 -C 8 )substituted heteroalkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, or R 5  together with R 6  form a five- or six-membered carbocyclic or heterocyclic ring or substituted heterocyclic ring; or R 4 , R 5  and R 6  together form fused carbocyclic or heterocyclic ring systems or substituted heterocyclic ring systems having five- or six-membered rings; 
 R 6  is selected from a group consisting of —H, -halogen, —CF 3 , —OH, —NH 2 ,—CCH, —CN, —NO 2 , (C 1 -C 8 )alkyl, (C 1 -C 8 )substituted alkyl, (C 1 -C 8 )alkylthio, (C 1 -C 8 ) substituted alkylthio, (C 1 -C 8 )alkoxy, (C 1 -C 8 )substituted alkoxy, —NR 7 R 8  where R 7  and R 8  are independently selected from H, (C 1 -C 8 )alkyl or R 7  and R 8  together form a four-, five-, six-, or seven-membered heterocycle, (C 1 -C 8 )heteroalkyl, (C 1 -C 8 )substituted heteroalkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl; 
 X is selected from —OH, —OR 11  and —OC(O)R 15 —; 
 R 11 , and R 15 , are independently selected from (C 1 -C 8 )alkyl, (C 1 -C 8 )substituted alkyl, —CH 2 CH(OH)CH 2 (OH), —CH(CH 2 OH) 2 , —CH 2 CCH, —(CH 2 CH2O)a—CH 2 CH 2 OH where a is 1, 2, 3, 4, 5, 6, 7, 8, 9 or  10 , —P(NR 22 R 23 )OCH 2 CH 2 -EWG, —P(O)(O − )-Oligonucleotide, —(CH 2 ) b CC-Nucleoside, —(CH 2 ) b CC -[Nucleoside Triphosphate], —(CH 2 ) b -Nucleoside, —(CH 2 ) b -[Nucleoside Triphosphate], —(CH 2 ) b NH-Nucleoside, —(CH 2 ) b NH-[Nucleoside Triphosphate], —(CH 2 ) b NHC(O)(CH 2 ) b -Nucleoside, —(CH 2 ) b NHC(O)(CH 2 ) b -[Nucleoside Triphosphate], where b is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; 
 EWG is an electron withdrawing group; and 
 R 22 , and R 23 , are independently selected from —CH 3 , —CH 2 CH 3 , —CH(CH 3 ) 2 , or R 22  and R 23  together form a heterocycle. 
 
       
     
     
       9. The compound according to  claim 8 , wherein R 4  is —H or R 4  together with R 5  form a five- or six-membered carbocyclic or heterocyclic ring. 
     
     
       10. The compound according to  claim 1 , wherein X is —OR 11  and R 11  represents —P(NR 22 R 23 )OCH 2 CH 2 -EWG, wherein 
       EWG is an electron withdrawing group and R 22 , and R 23  are independently selected from —CH 3 , —CH 2 CH 3 , —CH(CH 3 ) 2 , or R 22  and R 23  together form a heterocycle. 
     
     
       11. The compound according to  claim 1 , wherein the compound is of Structure 15. 
     
     
       12. The compound according to  claim 1 , wherein the compound is of Structure 16. 
     
     
       13. The compound according to  claim 1 , wherein the compound is of Structure 17. 
     
     
       14. The compound according to  claim 1 , wherein the compounds of structure 15 or 16, X is —NH 2 , —NHR 12 , —NR 13 R 14 , —NHC(O)R 16 . 
     
     
       15. The compound according to  claim 1 , wherein, in the compound of structure 16, R 7  and R 8  are independently selected from H or (C 1 -C 8 )alkyl. 
     
     
       16. The compound according to  claim 1 , wherein, in the compound of structure 17, R 24  and R 25  are (C 1 -C 8 )alkyl. 
     
     
       17. A compound of Structure 27, 28 or 29 
       
         
           
           
               
               
           
         
         where
 m is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, 
 R 2  is selected from a group consisting of —H or -CH 3 ; 
 R 9  is —CH 3 , —CH 2 CH 3 , —CH(CH 3 ) 2 ; 
 X is —OH, —NH2, —SH, —OR 11  , —NHR 12 , —NR 13 R 14 , —OC(O)R 15 , —NHC(O)R 16 , —SC(O)R 17 , —C(O)OH, —C(O)OR 18 , —C(O)NHR 19 , —C(O)NR 20 R 21 , —N 3 ; 
 R 11 , R 12 , R 13 , R 14 , R 15 , R 16 , R 17 , R 18 , R 19 , R 20  and R 21  are independently selected from (C 1 -C 8 )alkyl, (C 1 -C 8 )substituted alkyl, —CH 2 CH(OH)CH 2 (OH), —CH(CH 2 OH) 2 , —CH 2 CCH, —(CH 2 CH 2 O) a -CH 2 CH 2 OH where a is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, —P(NR 22 R 23 )OCH 2 CH 2 -EWG, —P(O)(O—)-Oligonucleotide, —(CH 2 ) b CC-Nucleoside, —(CH 2 ) b CC-[Nucleoside Triphosphate], —(CH 2 ) b -Nucleoside, —(CH 2 ) b -[Nucleoside Triphosphate], —(CH 2 ) b NH-Nucleoside, —(CH 2 ) b NH-[Nucleoside Triphosphate], —(CH 2 ) b NHC(O)(CH2) b -Nucleoside, —(CH 2 ) b NHC(O)(CH 2 ) b -[Nucleoside Triphosphate], where b is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, 
 EWG is an electron withdrawing group, 
 R x  and R y  are independently H and CH 3 ; 
 R 22  and R 23  are independently selected from —CH 3 , —CH 2 CH 3 , —CH(CH 3 ) 2  or R 22  and R 23  together form a heterocycle; and 
 in the compound of Structure 27, R x  and R y  are independently H and CH 3 ; 
 in the compound of Structure 28, R 7  and R 8  are independently selected from H, (C 1 - C 8 )alkyl, (C 1 -C 8 )substituted alkyl, or R 7  and R 8  together form a five-, six-, or seven-membered heterocycle; and 
 in the compound of Structure 29, 
 R 24  is (C 1 -C 8 )alkyl or (C 1 -C 8 )substituted alkyl; 
 R 25  is (C 1 -C 8 )alkyl, (C 1 -C 8 )substituted alkyl or CH 2 SO 3 H; and 
 the dashed line represents an optional double bond. 
 
       
     
     
       18. The compound according to  claim 17 , wherein X is —OH, —OR 11 , or OC(O)R 15 . 
     
     
       19. The compound according to  claim 17 , wherein, in the compound of Structure 28, R 7  and R 8  are independently selected from H or (C 1 -C 8 )alkyl. 
     
     
       20. The compound according to  claim 17 , wherein, in the compound of Structure 29, R 24  and R 25  are (C 1 -C 8 )alkyl. 
     
     
       21. The compound according to  claim 17 , wherein X is —OR 11  and R 11  represents —P(NR 22 R 23 )OCH 2  CH 2  -EWG, wherein 
       EWG is an electron withdrawing group and R 22 , and R 23  are independently selected from —CH 3 , —CH 2 CH 3 , —CH(CH 3 ) 2 , or R 22  and R 23  together form a heterocycle. 
     
     
       22. The compound according to  claim 17 , wherein the compound is of Structure 27. 
     
     
       23. The compound according to  claim 17 , wherein the compound is of Structure 28. 
     
     
       24. The compound according to  claim 17 , wherein the compound is of Structure 29. 
     
     
       25. A coumarin-CPG compound of Structure 150, wherein the coumarin-CPG compound is of Structure 150, 
       
         
           
           
               
               
           
         
       
       wherein the coumarin moiety is of structure 153, 154 or 155, 
       
         
           
           
               
               
           
         
       
       wherein: 
       m is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; 
       R 2  is H or CH 3 ; 
       R 9  is CH 3 , CH 2 CH 3 or CH(CH 3 ) 2 ; 
       in the moiety of Structure 153, R x  and R y  are independently H or CH 3 ;
 in the moiety of Structure 154, R 7  and R 8  are independently selected from H, (C 1 -C 8 )alkyl, (C 1 -C 8 )substituted alkyl, or R 7  and R 8  together form a four-, five-, six-, or seven-membered heterocycle; 
 in the moiety of Structure 155, R 24  is (C 1 -C 8 )alkyl or (C 1 -C 8 )substituted alkyl, R 25  is (C 1 -C 8 )alkyl, (C 1 -C 8 )substituted alkyl or CH 2 SO 3 H and the dashed lines represents an optional double bond; 
 Z is (CH 2 ) x , wherein x is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; 
 Linker 1 is selected from OC(O)(CH 2 ) y O(CH 2 ) z (CO), NHC(O)(CH 2 ) y O(CH 2 ) z (CO), wherein y is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, and wherein z is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; and 
 Linker 2 is selected from NH(CH 2 ) a , wherein a is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.